The utilization of the NIH-3T3 cell transfection method has enabled the isolation of several novel oncogenes. The goal of this project is to investigate the mechanism of activation and function of the novel human lbc transforming gene obtained by NIH-3T3 transfection with human leukemic DNA and isolated by its ability to form tumors in nude mice. The lbc cDNA sequence shows no identity to known sequences, and encodes a predicted hydrophilic protein of approximately 47 kD. Lbc is expressed in human myeloid and lymphoid cells and muscle, lung and heart tissue. Transfection of NIH-3T3 cells with lbc cDNA results in morphologically transformed foci in culture, demonstrating the biological activity of the lbc cDNA, and presenting a functional in vitro assay for the analysis of lbc transforming activity. The following aims are proposed. A. Preliminary studies indicate that the lbc oncogene represents a novel transcription unit derived from fusion of a truncated lbc proto-oncogene located on chromosome 15 with heterologous sequence encoded on chromosome 7. In order to fully elucidate the mechanism of activation, it is proposed to obtain complete lbc proto-oncogene cDNA sequence from normal hematopoietic tissues, to analyze the transforming activity of the proto-oncogene and its mutant forms, and to precisely define the breakpoint by genomic sequence analysis. B. Human samples/cell lines from hematopoietic malignancies and disorders will be screened for activated lbc genes, based on lbc expression. The exact location of the lbc gene on human chromosome 15, and in the mouse will be pinpointed and compared with the position of genetic loci known to be involved in hematologic and other diseases in human and mouse. C. The transforming activity of lbc will be analyzed by focusing on two functionally implicated domains: 1. A dbl domain associated with regulatory activity for the ras superfamily of small G proteins which control cell proliferation and cytoskeletal organization; this domain is also encoded in the dbl and vav oncogenes, the bcr gene and the yeast cell cycle gene CDC24. 2. A PH (pleckstrin homology) domain found in several intracellular signal transducing molecules, thought to define a novel protein interaction domain. These domains will be subjected to site-directed mutagenesis, and mutants assayed using the 3T3 focus forming assay. D. In order to study lbc function, antibodies will be derived to identify the lbc protein, to determine its cellular localization and to analyze lbc phosphorylation. Based on the activity associated with the dbl domain encoded in other oncogenes, it is proposed to test already available recombinant lbc protein for regulatory activity against a panel of ras-like small G proteins. Furthermore, the role of lbc in the p2lras signal transduction pathway will be analyzed using a well- characterized dominant negative ras mutant Nl7. These proposed experiments will elucidate the transforming activity and function of the novel lbc oncogene.